Codon optimized Thermobifida fusca hydrolase secreted by Bacillus megaterium
Biochemical Engineering, Technical University Braunschweig, GBF/TU-BCE, Mascheroder Weg 1, 38124 Braunschweig, Germany. Biotechnology and Bioengineering
(Impact Factor: 4.13).
03/2007; 96(4):780-94. DOI: 10.1002/bit.21167
Production and secretion of a 28,172 Da hydrolase from Thermobifida fusca (TFH) in Bacillus megaterium MS941 and WH323 was investigated in shake flask and pH controlled bioreactors. Successful production of heterologous TFH was achieved by adapting the original tfh gene to the optimal codon usage of B. megaterium. A codon adaption index close to one was reached. The codon optimized tfh was cloned into an open reading frame with DNA sequence for the N-terminal signal peptide of B. megaterium lipase A and a C-terminal His(6)-tag, all under the control of a xylose inducible promoter. Successful TFH production and secretion were observed using batch reactor cultivations with complex medium. Expression of the tfh gene from the P(xylA) promoter and secretion of produced TFH were compared in detail to batch reactor cultivations with semi-defined growth medium. For the first time, significant TFH secretion was achieved using a semi-defined medium in glucose limited fed batch cultivations yielding 10-fold higher cell densities compared to LB medium cultivation. Comparable volumetric TFH activities were obtained for both cultivation strategies. Surprisingly, measured specific TFH activities exhibited drastic discrepancies between preparations from LB and semi-defined medium grown B. megaterium. TFH recovery by Ni-chelate affinity chromatography resulted in higher purification factors when LB medium was used. These results indicated that secreted TFH is favorably produced by batch cultures of B. megaterium WH323 in LB medium.
Available from: Ren Wei
- "E. coli BL21- Gold(DE3) pET- 20b(+) α- hemolysin secretion pathway Tfu_0883 (Su et al., 2012) Bacillus megaterium MS941 / WH323 pYYBm LipA of B. megaterium TfH C-terminal (Fürch et al., 2007; Yang et al., 2007 "
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ABSTRACT: Abstract Thermophilic actinomycetes produce enzymes capable of hydrolyzing synthetic polyesters such as polyethylene terephthalate (PET). In addition to carboxylesterases, which have hydrolytic activity predominantly against PET oligomers, esterases related to cutinases also hydrolyze synthetic polymers. The production of these enzymes by actinomycetes as well as their recombinant expression in heterologous hosts is described and their catalytic activity against polyester substrates is compared. Assays to analyze the enzymatic hydrolysis of synthetic polyesters are evaluated, and a kinetic model describing the enzymatic heterogeneous hydrolysis process is discussed. Structure鈥揻unction and structure鈥搒tability relationships of actinomycete polyester hydrolases are compared based on molecular dynamics simulations and recently solved protein structures. In addition, recent progress in enhancing their activity and thermal stability by random or site-directed mutagenesis is presented.
Available from: Zou Wei
- "Further, compared to Gram-negative organisms such as Escherichia coli, B. megaterium lacks endotoxins and does not synthesize alkaline proteases that are capable of degrading recombinant proteins, making it an excellent expression host for the production of recombinant proteins (Vary, 1992). Therefore, B. megaterium is commonly employed to produce different industrial enzymes including Thermobifida fucsa hydrolase (TFH) (Yang et al., 2007), penicillin G acylase (Marina Pinotti et al., 2007), and various amylases (Vihinen and Mantsala, 1989). In addition, the bacterium is used for pharmaceutical production of substances such as oxetanocin (Morita et al., 1999). "
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ABSTRACT: A genome-scale metabolic model of Bacillus megaterium WSH002, an industrial bacterium widely used in the vitamin C industry, was reconstructed on the basis of the genome annotation and data from the literature and biochemical databases. It comprises 1112 reactions, 993 metabolites, and 1055 genes, including 43 new annotated genes. This model was able to predict qualitatively and quantitatively the growth of B. megaterium on a range of carbon and nitrogen sources, and the results agreed well with experimental data. A gene essentiality analysis predicted a core metabolic essential gene set of 57 genes on three different media. Furthermore, constraint-based analysis revealed that B. megaterium WSH002 is capable of producing and exporting several key metabolites, which could promote the growth of Ketogulonicigenium vulgare and 2-keto-l-gulonic acid (2-KLG) production. Here, the model represents a helpful tool for understanding and exploring this important industrial organism.
- "During the last years, B. megaterium was developed for the high yield production, secretion, and purification of recombinant proteins by improving different elements of the used plasmid systems. This strategies included the introduction of signal peptides for secretion of recombinant proteins (Biedendieck et al. 2007a; Malten et al. 2006; Stammen et al. 2010a), the utilization of affinity tags for protein purification (Biedendieck et al. 2007c; Malten et al. 2006), the optimization of the established xylose-inducible promoter system (Korneli et al. 2013a; Stammen et al. 2010a), the development of new inducible promoter systems (Biedendieck et al. 2007b; Gamer et al. 2009; Stammen et al. 2010b), and the use of codon adapted genes (Bäumchen et al. 2007; Yang et al. 2007). In this last context, the adaptation of the codon usage to the host B. megaterium has to occur individually for each gene in a time-and cost-intensive manner. "
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